Genotypic and phenotypic analysis of zwittermicin A-producing strains of Bacillus cereus

Many strains of Bacillus cereus produce zwittermicin A, a novel antibiotic that contributes to the ability of B. cereus to suppress certain plant diseases. The purpose of this study was to identify molecular indicators of zwittermicin A production in B, cereus strains, contribute to an understanding of the ecology and evolution of this group of bacteria, and identify potential agents for control of plant disease. The fatty acid composition of 20 strains known to be zwittermicin A producers and 20 strains known to be non-producers was determined. Cluster analysis of the fatty acid methyl ester (FAME) profiles revealed that zwittermicin A producers grouped together in two clusters, apart from most non-producers. Discriminant analysis of the FAME profiles generated models that correctly predicted the zwittermicin A-production phenotype in 17 of 20 zwittermicin A producers and 17 of 20 non-producers. Sixteen random oligonucleotide primers were tested in PCR, and one primer was identified that generated a fragment of 0.48 kb or 0.49 kb from total DNA from 26 of 28 strains known to produce zwittermicin A, whereas PCR with this primer did not generate bands of that size from 16 of 20 non-producing strains. PCR with primers designed to amplify zmaR, a gene from B. cereus that confers resistance to zwittermicin A, generated DNA fragments of 1.1 kb and 1.0 kb in all 29 zwittermicin A-producing strains tested, amplified a fragment of 0.3 kb in some of the zwittermicin A-producing strains, and amplified no fragments in 20 of 23 non-producing strains in a stock collection of B. cereus strains. The zmaR primers were tested for their ability to identify new zwittermicin A-producing isolates of B. cereus from two soils. All 12 of the isolates that produced the banding pattern characteristic of this primer pair produced zwittermicin A, and none of the 12 isolates that did not have the banding pattern produced detectable zwittermicin A. Seven of the 12 isolates initially identified as zwittermicin A producers with the zmaR primers significantly suppressed damping-off of alfalfa, whereas only one of the non-producers suppressed this disease. The results show that FAME and PCR analyses distinguish B. cereus strains that produce zwittermicin A from other B. cereus strains, that PCR with the primers designed to amplify zmaR is the most reliable method of those tested for identification of zwittermicin A producers, and that this method can be used to identify new strains with disease-suppressive activity.